Methods for eliminating or reducing the expression of genes in filamentous fungal strains by transitive RNA interference

ABSTRACT

The present invention relates to methods of reducing or eliminating expression of a target gene in a filamentous fungal strain by transitive RNA interference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser. No. 12/520,067 filed Dec. 20, 2007, which is a 35 U.S.C. §371 national application of PCT/US2007/088447 filed on Dec. 20, 2007 and claims priority from U.S. Provisional Application Ser. No. 60/876,895 filed on Dec. 21, 2006, which applications are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods of reducing or eliminating expression of a gene in a filamentous fungal strain.

Description of the Related Art

Filamentous fungal strains are widely used for the production of biological substances of commercial value. However, filamentous fungal strains with desirable traits of increased expression and secretion of a biological substance may not necessarily have the most desirable characteristics for successful fermentation. The production of a biological substance may be accompanied by the production of other substances, e.g., enzymes, that degrade the biological substance or co-purify with the biological substance, which can complicate recovery and purification of the biological substance.

One solution to these problems is to inactivate the gene(s) involved in the production of the undesirable substance. Inactivation can be accomplished by deleting or disrupting the gene(s) using methods well known in the art. However, in some cases, inactivation of the gene may be difficult because of poor targeting to homologous regions of the genome. Inactivation can also be accomplished by random mutagenesis, which is not always specific for the intended target gene and other mutations are often introduced into the host organism. In other situations, the gene and its product may be required for survival of the filamentous fungal strain. Where multiple genes are to be inactivated by deletion or disruption, the task can be very cumbersome and time-consuming. When highly homologous members of gene families exist, deletion or disruption of all members can be extremely tedious and difficult.

In recent years various forms of epigenetic gene regulation have been described (Selker, 1997, Trends Genet. 13: 296-301; Matzke and Matzke, 1998, Cell. Mol. Life. Sci. 54: 94-103). These processes influence gene expression by modulating the levels of messenger RNA (Hammond and Baulcombe, 1996, Plant Mol. Biol. 32: 79-88; Xi-song Ke et al., 2003, Current Opinion in Chemical Biology 7: 516-523) via micro RNAs (Morel et al., 2000, Curr. Biol. 10: 1591-1594; Bailis and Forsburg, 2002, Genome Biol. 3, Reviews 1035; Grewal and Moazed, 2003, Science 301: 798-802).

Based on genetic studies of Drosophila and Caenorhabditis elegans, RNA interference (RNAi), also known as post-transcriptional gene silencing (in plants), is understood to involve silencing expression of a gene by assembly of a protein-RNA effector nuclease complex that targets homologous RNAs for degradation (Hannon, 2002, Nature 418: 244-251). The processing of double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) is accomplished by a family of enzymes known as Dicer (Bernstein et al., 2001, Nature 409: 363). Dicer, a member of the RNase III family of endonucleases that specifically cleaves dsRNA, is responsible for digestion of dsRNA into siRNAs ranging from 20-25 nucleotides (Elbashir et al., 2001, Nature 411: 494). These siRNAs then associate with the RNA Induced Silencing Complex (RISC) (Elbashir et al., 2001, Genes and Dev. 15: 188; NyKanen et al., 2001, Cell 197: 300; Hammond et al., 2001, Science 293: 1146). Although not well understood, RISC targets the mRNA from which the anti-sense fragment was derived followed by endo and exonuclease digestion of the mRNA effectively silencing expression of that gene. RNAi has been demonstrated in plants, nematodes, insects, mammals, and filamentous fungi (Matzke and Matzke, 1998, supra; Kennerdell et al., 2000, Nat. Biotechnol. 18: 896-8; Bosher et al., 1999, Genetics 153: 1245-56; Voorhoeve and Agami, 2003, Trends Biotechnol. 21: 2-4; McCaffrey et al., 2003, Nat. Biotechnol. 21: 639-44; WO 03/050288; WO 01/49844; WO 98/53083; and WO 05/056772).

Transitive RNAi, also known as spreading, refers to the movement of the silencing signal beyond a particular gene. In plants, transitive silencing has been found to occur both upstream and downstream of the mRNA targeted for gene silencing by double-stranded RNA (Fabian et al., 2002, Plant Cell 14: 857-867; Garcia-Perez et al., 2004, The Plant Journal 38: 594-602; Vaistij et al., 2002, The Plant Cell 14: 857-867; Van Houdt et al., 2003, Plant Physiol. 131: 245-253). In Caenorhabditis elegans, transitive RNAi has been described as silencing of the transcript upstream of the target dsRNA (Alder et al., 2003, RNA J. 9: 25-32; Hannon, 2002, Nature 418: 244-251; Sijen et al., 2001, Cell 107: 465-476). In C. elegans, descriptions of transitive RNAi indicate that in addition to siRNAs derived from the dsRNA target, secondary siRNAs sharing homology with 5′ flanking sequences are generated, presumably the result of RNA-dependent RNA polymerase (RdRP) and Dicer activity (Bleys et al., 2006, RNA J. 12: 1633-1639; Petersen et al., 2005, Plant Molecular Biology 58: 575-583). Transitive RNAi is not ubiquitous among insects and mammals (Chi et al., 2003, Proc. Natl. Acad. Sci. USA 100: 6343-6346; Hoa et al., 2003, Insect Biochemistry and Molecular Biology 33: 949-957; Roignamt et al., 2003, RNA J. 9:299-308).

Transitive RNAi differs from conventional RNAi in several ways. Although double-stranded RNA serves as inducer of both RNAi and transitive RNAi, transitive RNAi appears to require an RdRP, whereas RNAi alone does not. Consequently, in organisms demonstrating transitive RNAi, gene silencing is not limited by the boundaries of double-stranded RNA, and gene silencing can extend into flanking sequences. However, in organisms lacking transitive RNAi, gene silencing is confined within the region of double strandedness.

It would be an advantage in the art to have alternative methods of reducing or eliminating expression of one or more genes for strain development and improvement, functional genomics, and pathway engineering of filamentous fungal strains.

The present invention relates to methods of reducing or eliminating expression of one or more genes in a filamentous fungal strain.

SUMMARY OF THE INVENTION

The present invention relates to methods of reducing or eliminating expression of a target gene encoding a biological substance in a filamentous fungal strain, comprising:

(a) inserting into the genome of the filamentous fungal strain a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule; and

(b) inducing production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance.

The present invention also relates to a filamentous fungal strain comprising a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, is induced by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance.

The present invention further relates to methods of producing a biological substance of interest, comprising:

(a) cultivating a filamentous fungal strain under conditions conducive for production of the biological substance, wherein the filamentous fungal strain comprises a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to a target gene encoding an undesirable biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein RNA transcripts of the double-stranded transcribable nucleic acid construct are produced by the cultivating of the filamentous fungal strain that are then converted to short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the undesirable biological substance; and wherein the filamentous fungal strain comprises a third polynucleotide encoding the biological substance; and

(b) recovering the biological substance from the cultivation medium.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of transitive RNA interference in filamentous fungi.

FIG. 2 shows a restriction map of pCW098.

FIG. 3 shows a restriction map of pCW099.

FIG. 4 shows a restriction map of pEFer14.

FIG. 5 shows a restriction map of pDM261.

FIG. 6 shows a restriction map of pDM266.

FIG. 7 shows a restriction map of pAmFs031.

FIG. 8 shows a restriction map of pAL01.

FIG. 9 shows a restriction map of pAL02.

DEFINITIONS

Transitive RNA interference: The term “transitive RNA interference” or “transitive RNAi” is defined herein as the movement of the silencing signal beyond a particular gene. In transitive RNAi, double-stranded RNA (dsRNA) can serve as template for the synthesis of new dsRNA from which siRNA sharing homology with the target sequence results in the extension or spread of silencing of new sequence along the mRNA.

Short interfering RNAs: The term “short interfering RNAs” or “siRNAs” is defined herein as 20-25 nucleotide long RNA fragments, the products of Dicer mediated digestion of double-stranded RNA.

No effective homology: The term “no effective homology” is defined herein as corresponding nucleotides on a sense strand and the reverse complement strand, which comprises preferably less than 20, more preferably less than 15, even more preferably less than 10, and most preferably less than 5 contiguous nucleotides of identical sequence to the target gene.

Two segments complementary to each other in reverse orientation: The phrase “two segments complementary to each other in reverse orientation” is defined herein as one of several stretches or pieces of DNA that fit with others to constitute a whole and capable of undergoing Watson-Crick base pairing.

Transitive silenced target sequences: The phrase “transitive silenced target sequences” is defined herein as a dsRNA sequence earmarked for gene silencing, wherein the dsRNA sequence is the result of siRNA extension from adjoining sequences.

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment) cDNA: The term “cDNA” is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell (Sambrook, J., Fritsch, E. F., and Maniatis, T, 1989, Molecular Cloning: A Laboratory Manual. 2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). cDNA lacks intron sequences that are present in genomic DNA. The initial, primary RNA transcripts are precursor molecules that are processed through a series of steps before appearing as mature spliced mRNAs. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence.

Control sequence: The term “control sequences” is defined herein to include all components necessary for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

Promoter: The term “promoter” is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a biological substance to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of a coding region. The term “promoter” will also be understood to include the 5′ non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.

Mutant promoter: The term “mutant promoter” is defined herein as a promoter having a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides of a parent promoter, wherein the mutant promoter has more or less promoter activity than the corresponding parent promoter. The term “mutant promoter” also encompasses natural mutants and in vitro generated mutants obtained using methods well known in the art such as classical mutagenesis, site-directed mutagenesis, and DNA shuffling.

Hybrid promoter: The term “hybrid promoter” is defined herein as parts of two or more promoters that are fused together to generate a sequence that is a fusion of the two or more promoters, which when operably linked to a coding sequence mediates the transcription of the coding sequence into mRNA.

Tandem promoter: The term “tandem promoter” is defined herein as two or more promoter sequences, arranged in tandem, operably linked to a coding sequence for mediating transcription of the coding sequence into mRNA.

Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to additional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of reducing or eliminating expression of a target gene encoding a biological substance in a filamentous fungal strain, comprising: (a) inserting into the genome of the filamentous fungal strain a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule; and (b) inducing production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance.

FIG. 1 demonstrates transitive RNA interference. Transformants produce transcription products composed of a target segment flanked 3′ by an inverted repeat (IR) having no homology with the target gene. Double-stranded RNA (dsRNA) produced by folding and annealing of the IR are processed by Dicer producing siRNA sharing homology with the IR. Using the transcript as template, a portion of siRNAs are extended by an RNA-dependent RNA polymerase (RdRP) further than the 5′ IR boundary infiltrating the target sequence. Continued extension produces dsRNA encoded by the target, initiating RNAi.

The methods of the present invention provide new opportunities for strain development and improvement, functional genomics, and pathway engineering in filamentous fungal strains. For example, the present methods can be used as a tool for filamentous fungal host strain development by means of gene manipulation and pathway engineering or as replacement for gene knockouts, a time-consuming approach with variable rates of success. A gene may be resistant to inactivation by standard methods known in the art such as gene knockout. The methods of the present invention provide a solution for reducing or eliminating expression of such a gene. Gene knockouts are dependent on site-specific gene replacement. In fungi efficacy of this process is affected by chromosome locus, DNA sequences shared by the replacement construct and genome, and/or length of the shared homology. Attainment of transitive gene silencing as described is uniquely dependent on cloning of a portion of the target sequence upstream of a second sequence comprised of an inverted repeat. The methods are also particularly useful and efficient for reducing or eliminating a highly expressed gene in a particular filamentous fungal strain, which can be very important, for example, in developing the organism as a production host. This ability demonstrates the strength of the methods of the present invention. The methods are also useful for reducing or eliminating expression of a multiple of genes that are highly homologous to each other, especially genes of the same family or homologous genes in a biosynthetic or metabolic pathway. The methods are further useful because they can be manipulated to cause a variable reduction in expression of a biological substance. This variability is especially important where a complete knock-out of a gene encoding a biological substance would be lethal to a particular filamentous fungal strain, such as in a secondary pathway that feeds into a biosynthetic pathway of interest.

In the methods of the present invention, the first polynucleotide comprises a first transcribable region with homology to the target gene. The second polynucleotide comprises a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation.

The first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene may or may not be separated by a polynucleotide intervening sequence, which is a nucleotide sequence that has little or no homology to the first and second polynucleotides in the double-stranded transcribable nucleic acid construct. The polynucleotide sequences of the double-stranded transcribable nucleic acid construct may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

In a preferred aspect, the first and second polynucleotides are separated by a polynucleotide intervening sequence. The intervening sequence preferably consists of less than 150 nucleotides, more preferably less than 100 nucleotides, more preferably less than 60 nucleotides, more preferably less than 40 nucleotides, even more preferably less than 20 nucleotides, and most preferably less than 10 nucleotides.

In a more preferred aspect, the first and second polynucleotides are not separated by a polynucleotide intervening sequence.

The intervening sequence can be any nucleotide sequence without homology to the first or second polynucleotide and preferably has little or no homology to sequences in the genome of the filamentous fungal strain to minimize undesirable targeting/recombination.

Promoter

The promoter sequence may be native or foreign (heterologous) to the first homologous transcribable region and native or foreign to the filamentous fungal strain. In the methods of the present invention, the promoter can be a native promoter, heterologous promoter, mutant promoter, hybrid promoter, or tandem promoter.

Examples of promoters useful in the methods of the present invention include the promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

In preferred aspect, the promoter is the NA2-tpi promoter. In another preferred aspect, the promoter is the TAKA/NA2-tpi leader hybrid promoter.

Homologous Transcribable Region

The term “transcribable region with homology to a target gene” is defined herein as a nucleotide sequence that is homologous to the open reading frame of a target gene, or a portion thereof, and is transcribed into an RNA, e.g., ncRNA (non-coding RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), miRNA (micro RNA), or mRNA (messenger RNA), which may or may not be translated into a biological substance, e.g., polypeptide, when placed under the control of the appropriate regulatory sequences. The boundaries of the transcribable region are generally determined by the transcription start site located just upstream of the open reading frame at the 5′ end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3′ end of the mRNA. A homologous transcribable region can include, but is not limited to, genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences.

In the methods of the present invention, the transcribable region homologous to the target gene may be identical to the corresponding region of the target gene or may be a homologue thereof.

The degree of identity between the homologue and the corresponding region of the target gene required to achieve inactivation or reduction of expression of the target gene will likely depend on the target gene. The smaller the homologue's nucleotide sequence is relative to the entire target gene, the degree of identity between the sequences should preferably be very high or identical. The larger the homologue's nucleotide sequence is relative to the entire target gene, the degree of identity between the sequences can likely be lower.

In the methods of the present invention, the degree of identity of the homologue's nucleotide sequence to the corresponding region of the target gene is at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%. For purposes of the present invention, the degree of identity between two nucleic acid sequences is determined as defined herein.

Alternatively, the ability of the homologue and the corresponding region of the target gene to hybridize to each other under various stringency conditions can also provide an indication of the degree of relatedness required for inactivation or reduction of expression of a target gene. However, it should be recognized that the lower the stringency conditions required, e.g., low stringency, to achieve hybridization between the homologue and the corresponding region of the target gene, inactivation or reduction of expression of the target gene will likely be less efficient.

In a preferred aspect, the homologue and the corresponding region of the target gene hybridize under low stringency conditions. In a more preferred aspect, the homologue and the corresponding region of the target gene hybridize under medium stringency conditions. In an even more preferred aspect, the homologue and the corresponding region of the target gene hybridize under medium-high stringency conditions. In a most preferred aspect, the homologue and the corresponding region of the target gene hybridize under high stringency conditions. In an even most preferred aspect, the homologue and the corresponding region of the target gene hybridize under very high stringency conditions.

For probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency), more preferably at least at 50° C. (low stringency), more preferably at least at 55° C. (medium stringency), more preferably at least at 60° C. (medium-high stringency), even more preferably at least at 65° C. (high stringency), and most preferably at least at 70° C. (very high stringency).

For probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated T_(m) using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, lx Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.

For probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

The first homologous region preferably consists of at least 19 nucleotides, more preferably at least 40 nucleotides, more preferably at least 60 nucleotides, more preferably at least 80 nucleotides, even more preferably at least 100 nucleotides, and most preferably at least 200 nucleotides. The first homologous region can also consist of the entire open reading frame of the gene or a homologue thereof.

Non-Homologous Transcribable Region

The double-stranded transcribable nucleic acid construct also comprises a second transcribable region with no effective homology to the target gene or the host genome, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation.

In a preferred aspect, the second transcribable region is any transcribable part of any gene, such as the 5′-untranslated region, the coding sequence, or the 3′-untranslated region of a gene, which has no effective homology to the target gene or the host genome.

In a more preferred aspect, the second transcribable region corresponds to the coding sequence of a gene with no effective homology to the target gene or the host genome.

In another more preferred aspect, the second transcribable region corresponds to the 5′-untranslated region of a gene with no effective homology to the target gene or the host genome.

In another more preferred aspect, the second transcribable region corresponds to the 3′-untranslated region of a gene with no effective homology to the target gene or the host genome.

In a most preferred aspect, the second transcribable region is a portion of a non-endogenous gene, which has no homology to the target gene or the host genome, e.g., the hygromycin resistance gene of E. coli.

The second transcribable region preferably consists of at least 19 nucleotides, more preferably at least 40 nucleotides, more preferably at least 60 nucleotides, more preferably at least 80 nucleotides, more preferably at least 100 nucleotides, more preferably at least 250 nucleotides, even more preferably at least 500 nucleotides, most preferably at least 750 nucleotides, and even most preferably at least 1000 nucleotides.

The two segments complementary to each other in reverse orientation can be separated by a polynucleotide linker. The linker preferably consists of at least 4 nucleotides, more preferably of at least 20 nucleotides, more preferably at least 40 nucleotides, more preferably at least 60 nucleotides, more preferably at least 80 nucleotides, even more preferably at least 100 nucleotides, most preferably at least 250 nucleotides, and even most preferably at least 500 nucleotides.

Target Gene

The target gene may be any gene encoding a substance having a biological activity or any gene encoding a polypeptide having biological activity involved in the biosynthesis of a metabolite (hereinafter “biological substance”). The biological substance may be an RNA (e.g., ncRNA, rRNA, tRNA, miRNA, or mRNA). The biological substance may also be a polypeptide having a biological activity. The biological substance may also be a metabolite. The substance having biological activity may be native to the filamentous fungal strain or foreign or heterologous to the strain. A foreign or heterologous substance is a substance that is not native to the cell; or a native substance to which structural modifications have been made to alter the native substance.

In a preferred aspect, the biological substance is a polypeptide having biological activity. The polypeptide may be any polypeptide having a biological activity. The term “polypeptide” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, polypeptides and proteins. The term “polypeptide” also encompasses two or more polypeptides combined to form the encoded product. Polypeptides also include hybrid polypeptides, which comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the filamentous fungal cell. Polypeptides further include naturally occurring allelic and engineered variations of the above-mentioned polypeptides and hybrid polypeptides.

In a preferred aspect, the polypeptide is an antibody, antigen, antimicrobial peptide, enzyme, growth factor, hormone, immunodilator, neurotransmitter, receptor, reporter protein, structural protein, and transcription factor.

In a more preferred aspect, the polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In a most preferred aspect, the polypeptide is an acetylxylan esterase, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, ferulic acid esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, urokinase, or xylanase

In another preferred aspect, the polypeptide is an albumin, collagen, tropoelastin, elastin, or gelatin; or a variant or hybrid thereof.

The biological substance may also be the product of a selectable marker. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof.

It may be necessary in the practice of the present invention to isolate the target gene. The techniques used to isolate or clone a gene are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the gene from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application, Academic Press, New York. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the gene encoding a biological substance, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a filamentous fungal cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

In a preferred aspect, expression of the target gene is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, and even most preferably 100%.

Where it is desired to use a target sequence within the 5′ untranslated region, the coding sequence, or the 3′ untranslated region, gene silencing vectors constructed with inverted repeats within any one of these regions may additionally enable the silencing of genes that are homologous to the coding sequence present in the silencing vector. When it is, therefore, desired to silence homologues of a gene within an organism, the construction of a silencing vector containing a transitive expressed target sequence having homology within the 5′ untranslated region, the coding sequence, or the 3′ untranslated region may allow the elimination or reduction of expression of one or more genes exhibiting sequence homology to the coding sequence within the construct. The term “homology” or “homologous” usually denotes those sequences that are of some common ancestral structure and exhibit a high degree of sequence similarity of the active regions.

In a preferred aspect, the interfering RNA interacts with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene.

In a more preferred aspect, expression of one or more homologues of the target gene is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, and even most preferably 100%

Filamentous Fungal Strains

The present invention also relates to filamentous fungal strains comprising a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, is induced by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance.

The filamentous fungal strain may be any filamentous fungal strain useful in the methods of the present invention. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In a preferred aspect, the filamentous fungal strain is an an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma strain.

In a more preferred aspect, the filamentous fungal strain is an an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae strain. In another most preferred aspect, the filamentous fungal strain is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum strain. In another most preferred aspect, the filamentous fungal strain is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

In a most preferred aspect, the Aspergillus oryzae strain is Aspergillus oryzae strain deposit no. IFO 4177. In another most preferred aspect, the Fusarium venenatum strain is Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; as well as taxonomic equivalents of Fusarium venenatum regardless of the species name by which they are currently known. In another most preferred aspect, the Fusarium venenatum strain is a morphological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330. In another most preferred aspect, the Trichoderma reesei strain is Trichoderma reesei ATCC 56765. In another most preferred aspect, the Aspergillus niger strain is Aspergillus niger Bo-1 (DSM 12665). In another most preferred aspect, the Aspergillus niger strain is a mutant of Aspergillus niger Bo-1 (DSM 12665), as disclosed in WO 2004/090155.

Filamentous fungal strains may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma strains are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Reduction or elimination of expression of a target gene encoding an undesirable biological substance may be detected using methods known in the art that are specific for the targeted biological substance. These detection methods may include use of specific antibodies, high performance liquid chromatography, capillary electrophoresis, formation of an enzyme product, disappearance of an enzyme substrate, SDS-PAGE, or loss or appearance of a phenotype, e.g., spore color. For example, an enzyme assay may be used to determine the activity of the enzyme. Procedures for determining enzyme activity are known in the art for many enzymes (see, for example, D. Schomburg and M. Salzmann (eds.), Enzyme Handbook, Springer-Verlag, New York, 1990).

Methods of Production

The present invention also relates to methods of producing a biological substance of interest, comprising: (a) cultivating a filamentous fungal strain under conditions conducive for production of the biological substance of interest, wherein the filamentous fungal strain comprises a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to a target gene encoding an undesirable biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein RNA transcripts of the double-stranded transcribable nucleic acid construct are produced by cultivating the filamentous fungal strain under conditions to produce the RNA transcripts that are then converted to short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the undesirable biological substance; and wherein the filamentous fungal strain comprises a third polynucleotide encoding the biological substance of interest; and (b) recovering the biological substance of interest from the cultivation medium.

The biological substance of interest may be any biological substance as described herein. In a preferred aspect, the biological substance of interest is a polypeptide having biological activity. It may be native or foreign to the filamentous fungal strain. The reduction or elimination of expression of the target gene encoding the undesirable biological substance can lead to increased expression of another biological substance of interest. The undesirable biological substance could directly affect production or expression of the biological substance of interest. For example, the undesirable biological substance may be a protease that attacks the biological substance of interest thereby lowering the amount of the biological substance of interest produced. By reducing or eliminating expression of the protease, more of the biological substance of interest will be expressed and produced. Or, the undesirable biological substance may share a cellular process or processes, e.g., transcription factor or secretory pathway, with the biological substance of interest thereby lowering the amount of the biological substance of interest produced. By reducing or eliminating expression of the undesirable biological substance, more of the cellular process or processes will be available to the biological substance of interest, e.g., expression-limiting transcription elements, thereby increasing the amount of the biological substance of interest expressed and produced. Moreover, the undesirable biological substance may be a toxin that contaminates the biological substance of interest preventing the use of the biological substance of interest in a particular application, e.g., an enzyme in a food process.

In the production methods of the present invention, the filamentous fungal strains are cultivated in a nutrient medium suitable for production of the biological substance of interest using methods known in the art. For example, the strain may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the biological substance to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the biological substance is secreted into the nutrient medium, it can be recovered directly from the medium. If the biological substance is not secreted, it can be recovered from cell lysates.

The biological substance of interest may be detected using methods known in the art that are specific for the biological substances. These detection methods may include use of specific antibodies, high performance liquid chromatography, capillary chromatography, formation of an enzyme product, disappearance of an enzyme substrate, or SDS-PAGE. For example, an enzyme assay may be used to determine the activity of the enzyme. Procedures for determining enzyme activity are known in the art for many enzymes (see, for example, D. Schomburg and M. Salzmann (eds.), Enzyme Handbook, Springer-Verlag, New York, 1990).

The resulting biological substance of interest may be isolated using methods known in the art. For example, a polypeptide of interest may be isolated from the cultivation medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). A metabolite of interest may be isolated from a cultivation medium by, for example, extraction, precipitation, or differential solubility, or any method known in the art. The isolated metabolite may then be further purified using methods suitable for metabolites.

Polynucleotides Encoding a Biological Substance

An isolated polynucleotide sequence encoding a biological substance of interest may be obtained from any prokaryotic, eukaryotic, or other source. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the biological substance is produced by the source or by a cell in which a gene from the source has been inserted.

The techniques used to isolate or clone a polynucleotide encoding a biological substance of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotide from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application, Academic Press, New York. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the polynucleotide encoding the biological substance, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into the mutant filamentous fungal cell where multiple copies or clones of the nucleic acid sequence will be replicated. The polynucleotide may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Nucleic Acid Constructs

An isolated polynucleotide encoding a biological substance of interest may be contained in a nucleic acid construct in the filamentous fungal strain. A nucleic acid construct comprises a nucleotide sequence encoding the biological substance of interest operably linked to at least one promoter and one or more control sequences that direct expression of the nucleotide sequence in a filamentous fungal strain under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the biological substance of interest including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The isolated polynucleotide encoding the biological substance of interest may be further manipulated in a variety of ways to provide for expression of the biological substance. Manipulation of the nucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing recombinant DNA methods are well known in the art.

The polynucleotide may comprise one or more native control sequences or one or more of the native control sequences may be replaced with one or more control sequences foreign to the nucleotide sequence for improving expression of the coding sequence in a host cell. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a biological substance of interest.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding biological substance. The promoter sequence contains transcriptional control sequences that mediate expression of the biological substance. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

The control sequence may be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the biological substance. Any terminator that is functional in the filamentous fungal strain of choice may be used in the present invention.

Preferred terminators for filamentous fungal strain are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the filamentous fungal strain. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the biological substance. Any leader sequence that is functional in the filamentous fungal strain of choice may be used in the present invention.

Preferred leaders for filamentous fungal strains are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus nidulans triose phosphate isomerase, Fusarium venenatum trypsin, and Fusarium venenatum glucoamylase.

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence that, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the filamentous fungal strain of choice may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal strains are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a fungal host cell of choice may be used in the present invention.

Effective signal peptide coding regions for filamentous fungal strains are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow the regulation of expression of the biological substance relative to the growth of the filamentous fungal strain. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, Aspergillus oryzae glucoamylase promoter, and Fusarium venenatum glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the biological substance of interest would be operably linked with the regulatory sequence.

Expression Vectors

A polynucleotide encoding a biological substance of interest may be contained in a recombinant expression vector comprising a promoter, the nucleotide sequence encoding the biological substance, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vectors preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES Media and Solutions

amdS Overlay agar was composed per liter of 20 ml of COVE salt solution, 273.8 g of sucrose, 8 g of Noble agar, 10 mM acetamide, and 15 mM CsCl, pH 5.0.

AMG trace metals solution was composed per liter of 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3.0 g of citric acid.

Cellulase-inducing medium was composed per liter of 20 g of Arbocel-natural cellulose fibers (J. Rettenmaier USA LP), 10 g of corn steep solids (Sigma Chemical Co., St. Louis, Mo., USA), 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂, 0.42 g of MgSO₄.7H₂O, 0.42 ml of Trichoderma reesei trace metals solution, and 2 drops of pluronic acid. The pH was adjusted to 6.0 with 10 N NaOH before autoclaving.

COVE selection plates were composed per liter of 342.3 g of sucrose, 20 ml of COVE salt solution, 10 mM acetamide, 15 mM CsCl₂, and 25 g or 30 g of Noble agar.

COVE2 plates were composed per liter of 30 g of sucrose, 20 ml of COVE salt solution, 10 mM acetamide, and 25 g or 30 g of Noble agar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g of MgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE trace metals solution was composed per liter of 0.04 g of NaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g or 1 g of MnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

COVE A salt solution was composed per liter of 26 g of KCl, 26 g of MgSO₄, 76 g of KH₂PO₄, and 50 ml of COVE A trace elements solution.

COVE A trace elements solution was composed per liter of 0.04 g of NaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 0.8 g of FeSO₄.7H₂O, 0.8 g of MnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and 10 g of citric acid.

COVE A minus urea plus acetamide selective plates were composed per liter of 20 ml of COVE A salt solution, 220 g of sorbitol, 10 g of glucose, 10 mM acetamide, and 30 g of Bacto agar, pH 5.2.

M410 was composed per liter of 50 g of maltose, 50 g of glucose, 2 g of MgSO₄.7H₂O, 2 g of KH₂PO₄, 4 g of citric acid, 8 g of yeast extract, 2 g of urea, 0.5 ml of AMG trace metals solution, and 0.5 g of CaCl₂, pH 6.0.

Minimal medium was composed per liter of 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 10 g of glucose, 0.5 g of MgSO₄.7H₂O, and 0.004 g of D-biotin.

Minimal medium agar was composed per liter of 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Agar Noble, 10 g of glucose, 0.5 g of MgSO₄.7H₂O, and 0.004 g of D-biotin.

PDA plates were composed per liter of 39 g of DIFCO™ Potato Dextrose Sugar (Becton Dickinson and Co., Sparks, Md., USA).

PEG was composed of 60% PEG 4000 (Polysciences, Inc., Warrington, Pa., USA), 10 mM CaCl₂, and 10 mM Tris-HCl, pH 6.5, filter sterilized.

SPC was composed of 40% PEG 4000, 50 mM CaCl₂, and 0.8 M sorbitol, pH 4.5-5.5, filter sterilized.

20×SSC was composed per liter of 175.3 g of NaCl and 88.2 g of sodium citrate pH 7.0.

0.5×SSC was composed per liter of 4.38 g of NaCl and 2.2 g of sodium citrate pH 7.0.

STC was composed of 1 M sorbitol, 10 mM CaCl₂, and 10 mM Tris-HCl, pH 6.5, filter sterilized.

Trichoderma reesei trace metals solution was composed per liter of 216 g of FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid.

YPG was composed per liter of 10 g of yeast extract (Fisher Scientific, Fair Lawn, N.J., USA), 20 g of BACTO™ peptone (Becton Dickinson and Co., Sparks, Md., USA), and 20 g of glucose.

Example 1 Construction of Plasmid pCW098

Plasmid pCW098 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter (U.S. Pat. No. 6,461,837), an inverted repeat (hyg IR) made from a portion of the Escherichia coli hygromycin resistance gene (Kaster et al., 1983, Nucleic Acids Res. 11: 6895-6911), the Aspergillus niger amyloglucosidase (AMG) terminator (Hata et al., 1991, Agric. Biol. Chem. 55: 941-949, and the Aspergillus nidulans pyrG gene as a selectable marker (Ballance and Turner, 1985, Gene 36: 321-331).

To express a double-stranded RNA (dsRNA) derived from the Escherichia coli aph(4) (hygB) gene (Kaster et al., 1983, supra), one half of the inverted repeat, 199 base pairs from within the open reading frame of the aph(4) gene, was PCR amplified using a sense strand primer possessing a Not I restriction site and an antisense primer possessing a 5′ Sma I or Xma I restriction site, as shown below.

Primer cwhygnot.1 (sense): (SEQ ID NO: 1) 5′-gcggccgcGCGATGTTCGGGGATTCCCAATACGAGGTC-3′ Primer cwhygsma.1a (antisense): (SEQ ID NO: 2) 5′-cccgggGCATCATCGAAATTGCCGTCAACCAAGCTC-3′ Portions of the hygB coding sequence are shown in uppercase letters.

The amplification reaction (50 μl) was composed of 1× THERMOPOL™ Reaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.4 mM dNTPs, 100 ng of pSMai155 (WO 05/074647), 50 pmoles of sense primer, 50 pmoles of antisense primer, and 5 units of Taq DNA polymerase (New England Biolabs, Beverly, Mass., USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf AG, Hamburg, Germany) programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. (7 minute final extension).

A PCR product of 214 bp was purified by 1% agarose gel electrophoresis in TAE buffer (4.84 g of Tris Base, 1.14 ml of glacial acetic acid, and 2 ml of 0.5 M EDTA pH 8.0 per liter) and further purified using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA). The 214 bp PCR product was ligated with pCR2.1-TOPO® using a TOPO® TA Cloning Kit and transformed into ONE SHOT® TOP10 chemically competent E. coli cells according to the manufacturer's instructions (Invitrogen Corporation, Carlsbad, Calif., USA). Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA) and analyzed by DNA sequencing to identify those containing the desired hygB insert. One plasmid with the expected DNA sequence was designated MP#3.

The other half of the inverted repeat including a 100 base pair spacer was amplified using a sense strand primer possessing a 5′ Pac I restriction site and an antisense primer possessing a 5′ Sma I or Xma I restriction site shown below; hygB sequence is shown in uppercase letters.

Primer cwhygpac.2 (sense): (SEQ ID NO: 3) 5′-ttaattaaGCGATGTTCGGGGATTCCCAATACGAGGTC-3′ Primer cwhygsma.2a (antisense): (SEQ ID NO: 4) 5′-cccgggATCGGTCCAGACGGCCGCGCTTCTGCGGGC-3′

The amplification reaction (50 μl) was composed of 1× THERMOPOL™ Reaction Buffer, 0.4 mM dNTPs, 100 ng of pSMai155, 50 pmoles of sense primer, 50 pmoles of antisense primer, and 5 units of Taq DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. (7 minute final extension).

A PCR product of 314 bp was purified by 1% agarose gel electrophoresis in TAE buffer and further purified using a QIAQUICK® Gel Extraction Kit. The 314 bp PCR product was ligated with pCR2.1-TOPO® using a TOPO® TA Cloning Kit and transformed into ONE SHOT® TOP10 chemically competent E. coli cells according to the manufacturer's instructions. Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 and analyzed by DNA sequencing to identify those containing the desired hygB insert. One plasmid with the expected DNA sequence was designated MP#9.

MP#3 was digested with Not I and Xma I. MP#9 was digested with Pac I and Xma I. Both hygB DNA fragments were purified by 1% agarose gel electrophoresis in TAE buffer and extracted using a QIAQUICK® Gel Extraction Kit. The fragments were ligated to Not I/Pac I digested vector pAlLo2 (WO 05/056772, Example 21) to create pCW098 (FIG. 2).

Example 2 Construction of Plasmid pCW099

Plasmid pCW099 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter, a fragment of the Aspergillus oryzae wA gene (SEQ ID NO: 5 for the full-length genomic DNA sequence and SEQ ID NO: 6 for the deduced amino acid sequence), the Escherichia coli hygB inverted repeat (hyg IR), the Aspergillus niger amyloglucosidase (AMG) terminator, and the Aspergillus nidulans pyrG gene as a selectable marker.

A 176 bp fragment of the Aspergillus oryzae wA gene (SEQ ID NO: 7 for the DNA sequence and SEQ ID NO: 8 for the deduced amino acid sequence thereof) was PCR amplified from plasmid plus wA flanking sequence rescued from Aspergillus oryzae strain P2-5.1 (WO 05/056772, Examples 27 and 28) using primers cwwanco.1corr (sense) and cwwanot.1a (antisense) shown below. The sense primer was engineered to have a Nco I site at the 5′-end and the antisense primer was engineered to have a Not I site at the 5′-end.

Primer cwwanco.1corr (sense): (SEQ ID NO: 9) 5′-ccatggAGCACTTCGATTGCATTAG-3′ Primer cwwanot.1a (antisense): (SEQ ID NO: 10) 5′-gcggccgcAGAACGAACGCAGGTTTTATAC-3′ wA sequence is shown in uppercase letters.

The amplification reactions (50 μl) were composed of 1× THERMOPOL™ Reaction Buffer, 0.4 mM dNTPs, 100 ng of Aspergillus oryzae P2-5.1 DNA (WO 05/056772), 50 pmoles of primer cwwanco.1corr, 50 pmoles of primer cwwanot.1a, and 5 units of Taq DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. (7 minute final extension).

A PCR product of 188 bp was purified by 1% agarose gel electrophoresis in TAE buffer and further purified using a QIAQUICK® Gel Extraction Kit. The 188 bp PCR product was ligated with pCR2.1-TOPO® using a TOPO® TA Cloning Kit and transformed into ONE SHOT® TOP10 chemically competent E. coli cells according to the manufacturer's instructions. Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 and analyzed by DNA sequencing to identify those containing the desired wA insert. One plasmid with the expected DNA sequence was designated MP#10.

MP#10 DNA was digested with Nco I and Not I and purified by 1% agarose gel electrophoresis in TAE buffer. The wA fragment was extracted using a QIAQUICK® Gel Extraction Kit. The purified fragment was ligated to Nco I/Not I digested pCW098 to create pCW099 (FIG. 3).

Example 3 Construction of Plasmid pEFer14

Plasmid pEFer14 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter, a 176 bp fragment of the Aspergillus oryzae wA gene, the Escherichia coli hygB inverted repeat (hyg IR), the Aspergillus niger amyloglucosidase (AMG) terminator, and the full-length Aspergillus nidulans amdS gene as a selectable marker.

Plasmid pCW099 digested with Nco I and Pac I was purified by 1% agarose gel electrophoresis in TAE buffer. A 698 bp fragment containing the 176 bp wA fragment, and hygB inverted repeat was extracted using an ULTRAFREE®-DA column (Millipore, Billerica, Mass., USA) according to the manufacturer's instructions. The purified fragment was ligated to Nco I/Pac I digested pAlLo1 (WO 05/056772, Example 1) to create pEFer14 (FIG. 4).

Example 4 Construction of Plasmid pDM261

Plasmid pDM261 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter, the Escherichia coli hygB inverted repeat (hyg IR), the Aspergillus niger amyloglucosidase (AMG) terminator, and the full-length Aspergillus nidulans amdS gene as a selectable marker.

Plasmid pCW098 digested with Nco I and Pac I was purified by 1% agarose gel electrophoresis in TAE buffer. The 527 bp fragment containing the hygB inverted repeat was extracted using an ULTRAFREE®-DA column. The purified fragment was ligated to Nco I/Pac I digested pAlLo1 to create pDM261 (FIG. 5).

Example 5 Construction of Plasmid pDM266

Plasmid pDM266 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter, a 499 bp fragment of the Aspergillus oryzae wA gene, the Escherichia coli hygB inverted repeat (hyg IR), the Aspergillus niger amyloglucosidase (AMG) terminator, and the full-length Aspergillus nidulans amdS gene as a selectable marker.

A 499 bp fragment of the wA gene was amplified from Aspergillus oryzae strain A1560 (IFO 4177) genomic DNA using primers wA500FWD (sense) and wA500REV (antisense) shown below. The sense primer was engineered to have a Nco I site at the 5′-end and the antisense primer was engineered to have a Not I site at the 5′-end. Aspergillus oryzae strain A1560 (IFO 4177) genomic DNA was prepared using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions.

Primer wA500FWD (sense): (SEQ ID NO: 11) 5′-ccatggGCGCTCAAAAACAACATCAAC-3′ Primer wAREV (antisense): (SEQ ID NO: 12) 5′-gcggccgcAGAACGAACGCAGGTTTTAT-3′ wA sequence is shown in uppercase letters.

The amplification reactions (50 μl) were composed of 1× THERMOPOL™ Reaction Buffer, 0.2 mM dNTPs, 100 ng of Aspergillus oryzae strain A1560 genomic DNA, 50 pmoles of primer wA500FWD, 50 pmoles of primer wA500REV, and 2.5 units of Taq DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 30 seconds at 72° C. (10 minute final extension).

A PCR product of 513 bp was purified by 1% agarose gel electrophoresis in TAE buffer and further purified using an ULTRAFREE®-DA column. The wA fragment was ligated with pCR2.1-TOPO® using a TOPO® TA Cloning Kit and transformed into ONE SHOT® TOP10 chemically competent E. coli cells according to the manufacturer's instructions. Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 and analyzed by DNA sequencing to identify those containing the desired wA insert. One plasmid with the expected DNA sequence was designated MP#8.

MP#8 DNA was digested with Nco I and Not I and purified by 1% agarose gel electrophoresis in TAE buffer. The wA fragment was extracted using an ULTRAFREE®-DA column. The purified fragment was ligated to Nco I/Not I digested pDM261 to create pDM266 (FIG. 6).

The 499 bp wA fragment in pDM266 (basepairs 2607-3106 of the wA gene) has the same 3′ end as the 176 bp wA fragment in pCW099 and pEFer14.

Example 6 Transformation of Aspergillus oryzae and Analysis of Transformants

Aspergillus oryzae strain JaL250 (WO 98/11203) was grown on a PDA plate supplemented with 20 mM uridine for 7 days at 34° C. Spores were collected by adding 5 ml of 0.01% TWEEN® 80 (Fisher Scientific, Fair Lawn, N.J., USA), scraping the surface of the plate using a sterile inoculating loop, and collecting the spore suspension with a 5 ml pipet. Approximately 2-5×10⁷ spores were added to 100 ml of YPG medium in a 500 ml shake flask and incubated 16-18 hours at 30-34° C. and 140 rpm. Mycelia were collected using a sterile 0.2 μm 500 ml EXPRESS® filter unit (Millipore, Billerica, Mass., USA). Mycelia were filtered away from the growth medium, and then washed twice with 100 ml of 0.7 M KCl. Mycelia were re-suspended in 20 ml of protoplasting solution [5 mg/ml GLUCANEX® (Novozymes A/S, Bagsværd, Denmark) plus 0.5 mg/ml chitinase (Sigma Chemical Co., St. Louis, Mo., USA) in 0.7 M KCl]. Mycelia were transferred to a 125 ml shake flask and incubated at 34° C., 80 rpm for 30-90 minutes. Protoplasts were poured through a sterile funnel lined with MIRACLOTH™ (Calbiochem, San Diego, Calif., USA) into a sterile 50 ml polypropylene tube. Protoplasts were centrifuged 20 minutes at 1,303×g at room temperature in a Sorvall RT6000D centrifuge. The supernatant was discarded and protoplasts were re-suspended in 20 ml of STC. Protoplasts were centrifuged as above and re-suspended in 20 ml of STC. A 20 μl aliquot was removed and diluted with STC. Protoplasts were counted using a hemocytometer. The protoplasts were centrifuged as described above and re-suspended in the appropriate volume of STC to yield 2×10⁷ protoplasts/ml.

Five μg of pEFer14, pDM261, or pDM266 DNA were added to 100 μl of Aspergillus oryzae JaL250 protoplasts. After incubation at room temperature for 30 minutes, the protoplast/DNA mix was adjusted to 9 ml with STC, split into 3 aliquots, and spread onto three 150 mm COVE plates supplemented with 20 mM uridine and 1% maltose. The plates were then incubated at 34° C. Growth on acetamide required expression of the amdS gene present on each expression plasmid.

After 4 days of incubation, before mature spore coloration was apparent, 25 or 30 primary transformants obtained using plasmids pEFer14, pDM261, and pDM266 were streaked onto COVE2 plates supplemented with 20 mM uridine and 1% maltose. All colonies derived from transformants of pDM261 were uniformly dark green. In contrast, colonies obtained from transformants of pEFer14 or pDM266 varied in spore color ranging from light yellow to dark green. The transformants were purified by streaking spores to COVE2 plates supplemented with 20 mM uridine and 1% maltose and then picking isolated colonies to plates of the same medium. All plates were incubated at 34° C.

The results are shown in Table 1. Forty percent of the 30 spore purified pDM266 (499 bp wA) transformants showed spore coloration that was lighter than the wild-type. Thirty percent of the 30 spore purified pEFer14 (176 bp wA) transformants showed spore coloration that was lighter than wild-type. One hundred percent of the 25 spore purified pDM261 (no wA) transformants showed wild-type spore coloration.

TABLE 1 Transcribable % light % # Plasmid region colored wildtype screened pEFer14 176 bp wA, hyg IR 30 70 30 pDM266 499 bp wA, hyg IR 40 60 30 pDM261 hyg IR 0 100 25

The results showing light spore coloration of the pDM266 and pEFer14 transformants indicated a phenotype consistent with suppression of the wA gene by transitive RNAi.

Example 7 Southern Blot Analysis of wA Gene Silenced Aspergillus Oryzae Transformants

Southern blot analysis was performed to verify that varying spore coloration in six selected transformants from Example 6, as listed below in Table 2, were not the result of gene disruptions. Mutant Aspergillus oryzae P2-5.1 (WO 2005/056772) contained a disruption of the wA gene.

TABLE 2 Strain Name Spore Color JaL250 (untransformed) Wild-type DLM1610-45-pDM261#2 Wild-type P2-5.1 white DLM1641-74-pEFer14#3 light colored DLM1641-74-pDM266#17 white DLM1641-74-pDM266#24 Light colored DLM1641-74-pDM266#29 white

Genomic DNA from each of the above Aspergillus oryzae strains was prepared using a DNEASY® Plant Maxi Kit according to the manufacturer's instructions. Two μg of each genomic DNA were digested with Sap I and Cla I overnight at 37° C. Digested genomic DNA was fractionated by 0.7% agarose gel electrophoresis in TAE buffer for 17 hours and blotted onto a NYTRAN® SuPerCharged membrane (Schleicher & Schuell BioScience, Keene, N.H., USA) using a TURBOBLOTTER™ (Schleicher & Schuell BioScience, Keene, N.H., USA) for 14-16 hours, following the manufacturer's recommendations.

The membrane was first hybridized with a 463 bp digoxigenin-labeled Aspergillus oryzae wA probe, which was synthesized by incorporation of digoxigenin-11-dUTP by PCR using primer wA5primeFWD (sense) and primer wA5primeREV (antisense) shown below:

Primer wA5primeFWD (sense): 5′-TACTACGGAGACCTTGGAAA-3′ (SEQ ID NO: 13) Primer wA5primeREV (antisense): 5′-GCTCTTAGACAGCCTAGAAT-3′ (SEQ ID NO: 14)

The amplification reaction (50 μl) was composed of 1× THERMOPOL™ Reaction Buffer, 5 μl of PCR DIG Labeling Mix (Roche Applied Science, Indianapolis, Ind., USA), 10 ng of Aspergillus oryzae JaL250 genomic DNA (prepared using a DNEASY® Plant Maxi Kit), 10 pmol of primer wA5primeFWD, 10 pmol of primer wA5primeREV, and 2.5 units of Taq DNA polymerase. The reaction was incubated in an EPPENDORF® MASTERCYCLER® programmed for 30 cycles each for 30 seconds at 95° C., 30 seconds at 52° C., and 1 minute at 72° C. (7 minute final extension). The PCR reaction was purified by 0.8% agarose gel electrophoresis in TAE buffer where incorporation of digoxigenin was indicated by an increase in molecular mass. A 463 bp product band was excised from the gel and purified using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions.

Hybridization was performed in DIG Easy Hyb buffer (Roche Applied Science, Indianapolis, Ind., USA) at 42° C. for 15-17 hours. The membrane was then washed twice under high stringency conditions in 2×SSC plus 0.1% SDS for 5 minutes at room temperature followed by two washes in 0.5×SSC plus 0.1% SDS for 15 minutes each at 65° C. The probe-target hybrids were detected by a chemiluminescent assay (Roche Applied Science, Indianapolis, Ind., USA) following the manufacturer's instructions.

Southern blot analysis performed on the transformants revealed that in all transformants, hybridization bands were of the same size as that of Aspergillus oryzae host strain JaL250 containing the full-length wA gene compared to a significantly smaller hybridized band in P2-5.1. The results demonstrated that the non-wild-type spore colors were not the result of gene disruptions.

Example 8 Extraction of RNA from Aspergillus oryzae Transformants

Six Aspergillus oryzae transformants (Example 6) displaying varied spore colors were spread on COVE2 plates supplemented with 20 mM uridine and 1% maltose and cultivated for 7 days at 34° C. Spores of untransformed Aspergillus oryzae JaL250 were spread on PDA plates supplemented with 20 mM uridine and cultivated for 7 days at 34° C.

Following the addition of 5 ml of 0.01% TWEEN® 80, spores for each strain were collected by scraping the surface of the plate using a sterile disposable spreader (Arben Bioscience, Rochester, N.Y., USA). Each spore suspension was drawn up into a sterile 5 ml serological pipet, added to 75 ml of Minimal medium (pH 6.5) supplemented with 10 mM uridine and 1% maltose in a 500 ml shake flask, and cultivated for 22-24 hours at 34° C. and 65 rpm. A 47 mm cellulose nitrate filter (Whatman Inc., Florham Park, N.J., USA) was placed on top of a filter membrane in a sterile 0.2 μm 250 ml MF75 filter unit (Nalgene, Rochester, N.Y., USA). Twenty five to 75 ml of each whole culture broth were placed onto separate cellulose nitrate filters and subjected to vacuum filtration to produce a thin layer of mycelia on the filter. The cellulose nitrate filters plus mycelia were transferred to 60 mm Petri dishes containing Minimal medium agar (pH 6.5) supplemented with 10 mM uridine and 1% maltose. The agar plates were placed in a plastic bag, sealed, and incubated at 37° C.

When pigmented conidiaphores began to appear (42-48 hours) in the Aspergillus oryzae JaL250 control, 120 mg of mycelia were scraped from each filter using a spatula and each transferred to a Lysing Matrix C tube (Q-Biogene, Irvine, Calif., USA) containing 1 ml of RNAPRO™ Solution (Q-Biogene, Irvine, Calif., USA). The Lysing Matrix C tubes were securely capped to prevent leakage during homogenization. The sample tubes were processed for 40 seconds at speed 6 in a FASTPREP® FP120 Instrument (Q-Biogene, Irvine, Calif., USA), and then placed on ice for 2 minutes. The sample tubes were processed again for 40 seconds at speed 6, and then placed on ice for 2 minutes. The samples were centrifuged at 13,400×g in an EPPEN DORF® 5415D microcentrifuge for 5 minutes at 4° C. The resulting supernatants were transferred to 1.7 ml microcentrifuge tubes, and then incubated for 5 minutes at room temperature. Three-hundred μl of chloroform were added to the sample tubes and vortexed for 10 seconds. After incubation at room temperature for 5 minutes, the samples were centrifuged at 13,400×g in an EPPENDORF® 5415D microcentrifuge for 5 minutes at 4° C. The upper phase of each sample was transferred to a new 1.7 ml microcentrifuge tube, and then incubated for 5 minutes at room temperature. Again, 300 μl of chloroform were added to the samples and vortexed for 10 seconds. The samples were incubated for 5 minutes at room temperature, followed by centrifugation at 13,400×g in an EPPENDORF® 5415D microcentrifuge for 5 minutes at 4° C. The upper phases were transferred to new 1.7 ml microcentrifuge tubes. Five-hundred μl of ice cold ethanol was added to the sample tubes and incubated for 30 minutes at −20° C. The samples were centrifuged at 13,400×g in an EPPENDORF® 5415D microcentrifuge for 20 minutes at 4° C. The resulting supernatants were removed and the pellets washed with 75% ethanol. The ethanol was removed and the pellets were allowed to air-dry for 5 minutes at room temperature. The pellets were resuspended in 100 μl of diethylpyrocarbonate (DEPC)-treated water followed by the addition of 50 μl of 8 M lithium chloride (VWR, West Chester, Pa., USA). The samples were incubated for 1 hour at −20° C. After incubation, the samples were centrifuged at 13,400×g in an EPPENDORF® 5415D microcentrifuge for 25 minutes at 4° C. The supernatants were removed from the sample tubes, and the RNA pellets were rinsed with 70% ethanol. The ethanol was removed, and the RNA pellets were resuspended in 25-40 μl of DNase, RNase-free water (Sigma-Aldrich, St. Louis, Mo., USA). RNA concentration was quantified using a NANODROP® 1000 Spectrophotometer (Nanodrop Technologies, Wilmington, Del., USA).

Example 9 Treatment of Aspergillus oryzae RNA with DNase

Contaminating genomic DNA was removed from the Aspergillus oryzae RNA samples (Example 8) using a TURBO DNAfree™ Kit (Ambion, Austin, Tex., USA). Three μg of extracted RNA were combined with 1× TURBO DNase™ Buffer (Ambion, Austin, Tex., USA) and adjusted to a volume of 10 μl with DNase, RNase-free water. One unit of TURBO DNase™ (Ambion, Austin, Tex., USA) was added to the sample and incubated for 30 minutes at 37° C. Another 1 unit of TURBO DNase™ was added and the sample was incubated for 1 hour at 37° C. Two μl of DNase Inactivation Reagent (Ambion, Austin, Tex., USA) were added. The contents of the sample tubes were mixed 3 times during a 2 minute incubation period at room temperature to redisperse the DNase Inactivation Reagent. The DNase Inactivation Reagent was pelleted by centrifugation for 2 minutes at 9,300×g in an EPPENDORF® 5415D microcentrifuge. Nine μl of each supernatant was transferred to a 0.65 ml microcentrifuge tube. One μl of DNase-treated RNA was measured using a NANODROP® 1000 Spectrophotometer in order to determine RNA concentration.

Example 10 Synthesis of First Strand cDNA from Aspergillus oryzae Total RNA Samples

First strand cDNA was synthesized from each of the extracted Aspergillus oryzae RNA samples (Example 9) using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, Ind., USA). One μg of DNase-treated RNA was combined with 1.2 nmol of random hexamer primer (Roche Applied Science, Indianapolis, Ind., USA) and adjusted to a volume of 13 μl with DEPC-treated water. The sample was incubated for 10 minutes at 65° C., and then placed on ice. The 13 μl sample was combined with 1× Transcriptor RT Reaction Buffer (Roche Applied Science, Indianapolis, Ind., USA), 20 units of Protector RNase Inhibitor (Roche Applied Science, Indianapolis, Ind., USA), 10 mM deoxynucleotide mix, and 10 units of Transcriptor Reverse Transcriptase (Roche Applied Science, Indianapolis, Ind., USA) in a final volume of 20 μl. The reaction mixture was incubated for 10 minutes at 25° C., 60 minutes at 50° C., and 5 minutes at 85° C. After synthesis, the sample tubes were placed on ice.

To verify removal of contaminating genomic DNA and check integrity of the cDNA, the sample was PCR amplified with wA and actin primer sets that span introns shown below.

Primer 2wAFWD (sense): 5′-ATGCCTCGCAGCTTATAGGA-3′ (SEQ ID NO: 15) Primer 2wAREV (antisense): 5′-CGCACTGATATACGGTTTGG-3′ (SEQ ID NO: 16) Primer 2actinFWD (sense): 5′-GGATCTCTACGGTAACATCGTCA-3′ (SEQ ID NO: 17) Primer 2actinREV (antisense): 5′-GATCGGAGATGCCAGGGTA-3′ (SEQ ID NO: 18)

The amplification reactions (50 μl) were composed of 1× THERMOPOL™ Reaction Buffer, 0.4 mM dNTPs, 2 μl of template cDNA sample, 50 pmol of primer 2wAFWD, 50 pmol of primer 2wAREV, and 2.5 units of Taq DNA polymerase. A control amplification reaction (50 μl) was composed of 1× THERMOPOL™ Reaction Buffer, 0.4 mM dNTPs, 10 ng of Aspergillus oryzae JaL250 genomic DNA (prepared using a DNEASY® Plant Maxi Kit), 50 pmol of primer 2wAFWD, 50 pmol of primer 2wAREV, and 2.5 units of Taq DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER™ programmed for 30 cycles each for 30 seconds at 95° C., 30 seconds at 50° C., and 1 minute at 72° C. (7 minute final extension). The PCR reactions were purified by 0.8% agarose gel electrophoresis in TAE buffer. The results showed that PCR reactions using cDNA templates produced a smaller amplicon than the control PCR reactions using genomic DNA as templates. A PCR amplicon generated from genomic DNA will be larger than the corresponding amplicon generated from cDNA. Therefore, the cDNA preparations did not contain detectable amounts of genomic DNA.

Example 11 Detection of Steady-State wA mRNA Aspergillus oryzae by Real Time PCR (RT-PCR)

Determination of Aspergillus oryzae wA expression levels in the transformants was accomplished using RT-PCR. Complementary DNA (cDNA) synthesized from each transformant as described in Example 10 was used as a template for RT-PCR reactions. The wA gene served as the target DNA sequence, while the Aspergillus oryzae actin gene (SEQ ID NO: 19 for the DNA sequence and SEQ ID NO: 20 for the deduced amino acid sequence) served as the endogenous control, as well as the reference DNA sequence. Primers and their corresponding mono-color hydrolysis probes were selected and designed according to the Universal ProbeLibrary Assay Design Software Guide (Roche Applied Science, Indianapolis, Ind., USA). The probes were first selected based on the desired transcript DNA sequence near exon-exon splice junctions. The primer sets were then designed to amplify targets that span the same exon-exon splice junctions, or introns. The following probes were selected:

Universal ProbeLibrary Probe #131 (actin - reference) 5′-CTGGTGGT-3′ Universal ProbeLibrary Probe #134 (wA - target) 5′-CCTCCTTC-3′ The following primers were used: Primer 2wAFWD (sense): (SEQ ID NO: 21) 5′-ATGCCTCGCAGCTTATAGGA-3′ Primer 2wAREV (antisense): (SEQ ID NO: 22) 5′-CGCACTGATATACGGTTTGG-3′ Primer 2actinFWD (sense): (SEQ ID NO: 23) 5′-GGATCTCTACGGTAACATCGTCA-3′ Primer 2actinREV (antisense): (SEQ ID NO: 24) 5′-GATCGGAGATGCCAGGGTA-3′

The Relative Quantification Real-Time PCR assay was performed using a LIGHTCYCLER® 480 System (Roche Applied Science, Indianapolis, Ind., USA). Each RT-PCR reaction (20 μl) was composed of 1× LIGHTCYCLER® 480 Probes Master mix (Roche Applied Science, Indianapolis, Ind., USA), 200 nM sense primer, 200 nM antisense primer, 100 nM probe, and varying dilutions (i.e., undiluted, 1:10, 1:100, 1:1000) of cDNA template. The RT-PCR reactions were carried out in a LIGHTCYCLER® 480 System programmed for 45 cycles each for 10 seconds of denaturation at 95° C. and 30 seconds at 60° C. for both quantification and extension of amplified product. Each sample was prepared in triplicate using 384 well plates (Roche Applied Science, Indianapolis, Ind., USA). A negative control in which the template cDNA was replaced with PCR-grade water was run on every 384 well plate tested to reveal putative false-positive results.

Data obtained from the LIGHTCYCLER® 480 System was analyzed using LIGHTCYCLER® 480 Relative Quantification Software (Roche Applied Science, Indianapolis, Ind., USA). In the Relative Quantification method, each sample was corrected for differences in quality and quantity caused by discrepancies in initial sample concentration, variations in sample loading, pipetting errors, or disparities in cDNA synthesis efficiency by calculating the target gene concentration relative to a non-regulated reference gene. The target gene/reference gene ratio was not only determined, but also normalized by a calibrator that corrects for any differences in PCR efficiencies of the target and reference genes. The Relative Quantification analysis results were expressed as a normalized ratio in which the ratio of the target DNA sequence to the reference DNA sequence in a sample was divided by the ratio of the same two sequences in a standard sample or the calibrator.

A standard curve was first generated for both the target and reference using untransformed Aspergillus oryzae JaL250 as the standard sample. The standard curves yielded R² values of 0.996 and 0.995 for the target and reference, respectively. The PCR efficiency values calculated by the Roche LIGHTCYCLER® 480 Relative Quantification Software were 1.757 (error 0.0449) and 1.907 (error 0.0287) for the target and reference, respectively. In order to compare the relative expression levels of mRNA across all the strains listed in Example 7, real-time PCR reactions were carried out as described above. Data was collected and analyzed using the LIGHTCYCLER® 480 Relative Quantification Software and compared to the previously generated standard curves according to Roche LIGHTCYCLER® 480 Relative Quantification Software Manual (Roche Applied Science, Indianapolis, Ind., USA). Using Relative Quantification analysis, the expression level of wA mRNA in Aspergillus oryzae DLM1610-45-pDM261#2, the transformant with wild-type spore color, was comparable to the untransformed Aspergillus oryzae JaL250 strain. Relative expression levels of wA mRNA in Aspergillus oryzae DLM1641-74-pEFer14#3 and Aspergillus oryzae DLM1641-74-pDM266#24, transformants with light colored spores, showed a 67% and 62% reduction, respectively, as compared to the wA mRNA expression level in the strains with wild-type spore color. Aspergillus oryzae DLM1641-74-pDM266#17 and Aspergillus oryzae DLM1641-74-pDM266#29, strains with white spores, showed a 73% and 81% reduction in wA mRNA compared to the strains with wild-type spore color.

Example 12 Construction of Plasmid pAmFs031

A transitive RNAi expression vector was constructed for suppressing the expression of an Aspergillus niger ATCC 1015 polyketide synthase gene (SEQ ID NO: 25 for the cDNA sequence and SEQ ID NO: 26 for the deduced amino acid sequence).

Plasmid pAmFs031 was constructed to contain the TAKA/NA2-tpi leader hybrid promoter, a fragment of the open reading frame for the Aspergillus niger polyketide synthase gene, the Escherichia coli hygB inverted repeat (hyg IR), the Aspergillus niger amyloglucosidase terminator, and the full-length Aspergillus nidulans amdS gene (Kelly and Hynes, 1985, EMBO J. 4: 475-479) as a selectable marker. The Aspergillus niger polyketide synthase gene was selected as a silencing target because of its sequence identity to other polyketide synthases involved in conidial pigment biosynthesis in filamentous fungi.

A 502 base pair fragment from within the Aspergillus niger polyketide synthase open reading frame (SEQ ID NO: 27 for DNA sequence and SEQ ID NO: 28 for the deduced amino acid sequence) was amplified from genomic DNA isolated from Aspergillus niger strain MBin120 (WO 2004/090155) using a sense strand primer possessing a 5′ Nco I restriction site and an antisense primer possessing a 5′ Not I restriction site shown below.

Primer 56896F (sense): (SEQ ID NO: 29) 5′-ggggccatggTCAGCGCGGTAAGCTCTAAT-3′ Primer 56896R (antisense): (SEQ ID NO: 30) 5′-gggggcggccgcGTAAGGTTCCGCATTTCTGG-3′ Polyketide synthase coding sequence shown in uppercase letters.

The amplification reaction (50 μl) was composed of 1× HERCULASE® Reaction Buffer (Stratagene, La Jolla, Calif., USA), 0.2 mM dNTPs, 128 ng of Aspergillus niger MBin120 genomic DNA (prepared using a DNEASY® Plant Maxi Kit), 20 pmoles of sense primer, 20 pmoles of antisense primer, and 2.5 units of HERCULASE Hotstart DNA polymerase (Stratagene, La Jolla, Calif., USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. (7 minute final extension).

The resulting PCR product of 502 bp and pDM261 were digested with Nco I and Not I, purified by 0.75% agarose gel electrophoresis in TAE buffer, and further purified using a MINELUTE® Kit for the PCR product or a QIAQUICK® Gel Extraction Kit for the plasmid. The PCR product was ligated with the pDM261 plasmid fragment using a FAST-LINK™ DNA Ligation Kit (Epicentre Biotechnologies, Madison, Wis., USA) and transformed into SURE™ II chemically competent E. coli cells according to the manufacturer's instructions (Stratagene, La Jolla, Calif., USA). Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 and analyzed by DNA sequencing to identify those containing the desired polyketide synthase insert. One plasmid with the expected DNA sequence was designated pAmFs031 (FIG. 7).

Example 13 Transformation of Aspergillus niger and Analysis of Transformants

Aspergillus niger strain MBin120 was grown on a PDA plate for 14 days at 34° C. Spores were collected by adding 7 ml of 0.01% TWEEN® 20 (Fisher Scientific, Fair Lawn, N.J., USA), scraping the surface of the plate using a sterile inoculating loop, and collecting the spore suspension with a 5 ml pipet. A 500 ml glass shake flask containing 25 ml of YPG medium supplemented with 1 M sucrose was inoculated with 2.6×10⁸ spores and incubated 15 hours at 28° C. at 150 rpm. Mycelia were collected using a sterile 0.2 μm 500 ml EXPRESS® filter unit (Millipore, Billerica, Mass., USA). Mycelia were filtered from the growth medium, and then washed twice with 150 ml of 1 M sorbitol. Mycelia were resuspended in 30 ml of protoplasting solution containing 20 mg of GLUCANEX® and 0.4 mg of chitinase per ml of 1 M sorbitol. Mycelia were transferred to a 125 ml glass shake flask and incubated at 34° C., 100 rpm for 45 minutes. Protoplasts were poured through a sterile funnel lined with MIRACLOTH™ into a sterile 50 ml polypropylene tube. The tube was then filled with ice cold 1 M sorbitol. The protoplasts were centrifuged for 5 minutes at 1,303×g at room temperature in a Sorvall RT6000D centrifuge. The supernatant was discarded and the protoplasts were re-suspended in 50 ml of 1 M sorbitol. The protoplasts were centrifuged in a Sorvall RT6000D centrifuge and re-suspended in 10 ml of STC. A 20 μl aliquot was removed and diluted with STC. The protoplasts were counted using a hemocytometer. The protoplasts were centrifuged in a Sorvall RT6000D centrifuge and re-suspended in the appropriate volume of PEG 4000 (Polysciences, Inc., Warrington, Pa., USA) to yield 2×10⁷ protoplasts/ml.

Five μg of pAmFs031 or pDM261 DNA were added to 100 μl of the Aspergillus niger MBin120 protoplasts. After incubation on ice for 30 minutes, 1 ml of SPC was added to the protoplast/DNA solutions and gently mixed. The solutions were incubated for 30 minutes at room temperature. Then 10 ml of dissolved amdS overlay agar cooled to 50° C. was added to the transformation mixtures and spread onto 150 mm COVE plates. The plates were then incubated at 34° C. Growth on acetamide required expression of the amdS gene present on each expression plasmid.

After 4 days of incubation, 10 primary transformants were obtained for each of pAmFs031 and pDM261. The transformants were streaked onto COVE A minus urea plus acetamide plates supplemented with 1% maltose. All colonies derived from pDM261 were uniformly dark black. In contrast, the colonies obtained from the pAmFs031 transformants varied in spore color ranging from white to dark brown. The transformants were purified by streaking spores to COVE A minus urea plus acetamide plates supplemented with 1% maltose and then picking isolated colonies to plates of the same medium. Strain purification was repeated four times in total. All plates were incubated at 34° C.

Three of the 10 spore purified pAmFs031 transformants displayed spore coloration that was lighter than the wild-type. All of the 10 spore purified pDM261 “empty vector” transformants showed wild-type spore coloration. These results showing light spore coloration for the pAmFs031 transformants indicated a phenotype consistent with transitive RNAi associated suppression of the polyketide synthase gene.

Example 14 Growth and Total RNA Extraction from Polyketide Synthase Silenced and Control Aspergillus niger Strains

The following Aspergillus niger strains were grown for 7 days on COVE A minus urea plus acetamide plates supplemented with 1% maltose at 34° C.: two Aspergillus niger pAmFs031 strains with white spores (designated pAmFs031-W1 and pAmFs031-W2), one Aspergillus niger pAmFs031 strain with black spores (designated pAmFs031-B1), and one Aspergillus niger strain transformed with pDM261. The untransformed control strain Aspergillus niger MBin120 was grown on PDA plates. Spores were collected from each strain by adding 5 ml of 0.01% TWEEN® 20, scraping the surface of the plate with a sterile inoculating loop, and collecting the spore suspension with a 10 ml pipet. Forty μl of spore suspension were mixed with 10 ml of M410 medium and 1 ml of this mixture was added to each well of a 24 well polystyrene microtiter plate (Corning Incorporated, Corning, N.Y., USA). Each strain was grown in a separate microtiter plate to avoid cross contamination. The plates were incubated at 34° C. for 4 days in a humidity controlled chamber at which point the fungi formed a continuous mat of mycelial growth across the well and pigmented conidiaphores were present for all strains.

Total RNA was extracted from the mycelial mats using a RNAPRO™ Pro Red Kit (Q-Biogene, Irvine, Calif., USA). The mats were removed from the wells and placed onto a paper-towel to absorb any excess medium. Totally 200 mg of each mycelial mat tissue were transferred to a FASTRNA®ProRed tube containing 1 ml of RNAPRO™ solution (Q-Biogene, Irvine, Calif., USA). The mycelia were homogenized for 40 seconds at speed 6 using a FASTPREP® FP120 Instrument. Each sample was centrifuged at 13,400×g in a Sorvall MC12V microcentrifuge for 5 minutes at 4° C. The aqueous phases were transferred to 1.7 ml microfuge tubes. The samples were incubated at room temperature for 5 minutes. Three hundred μl of chloroform were added and the samples were vortexed 10 seconds and then incubated at room temperature for 5 minutes. The samples were centrifuged at 13,400×g in a Sorvall MC12V microcentrifuge for 5 minutes at 4° C. The upper phases were transferred to new 1.7 ml microfuge tubes. The samples were extracted again with chloroform as described above. Five hundred μl of ice cold ethanol were added and the samples were stored at −20° C. for one hour. The samples were centrifuged at 13,400×g in a Sorvall MC12V microcentrifuge for 20 minutes at 4° C. The ethanol was removed and the pellets were washed with 75% ethanol. The ethanol was removed and the pellets were air dried for 5 minutes. The RNA samples were resuspended in 100 μl of DEPC-treated water. Fifty μl of 8 M LiCl were added and the samples were stored for 1 hour at −20° C. The samples were centrifuged at 13,400×g in a Sorvall MC12V microcentrifuge for 25 minutes at 4° C. The LiCl was removed and 500 μl of 75% ethanol were added to the pellets and then removed. The pelleted samples were air dried for 5 minutes. The RNA samples were resuspended in 30 μl of DEPC-treated water. The RNA concentration was measured using a NANODROP® 1000 Spectrophotometer. Samples were stored at −80° C.

Example 15 First Strand cDNA Synthesis

RNA samples from each of the Aspergillus niger strains described in Example 14 were treated with DNase to remove genomic DNA using reagents from a TURBO DNase™ Kit (Ambion, Inc., Austin, Tex., USA). Three μg of RNA were combined with 1 μl of TURBO DNase™ buffer (Ambion, Inc., Austin, Tex., USA) and adjusted to a volume of 10 μl with DEPC-treated water. A 0.5 μl aliquot of TURBO DNase™ (Ambion, Inc., Austin, Tex., USA) was added and the sample was incubated 30 minutes at 37° C. A second 0.5 μl aliquot of TURBO DNase™ was added and the sample was again incubated for 30 minutes at 37° C. Two μl of Inactivation Reagent (Ambion, Inc., Austin, Tex., USA) were added and the sample was mixed three times during a 2 minute incubation at room temperature. The sample was centrifuged for 2 minutes at 9,300×g in an EPPENDORF® 5415D microcentrifuge. Nine μl of supernatant were transferred to a 0.6 ml microfuge tube. The RNA concentration was measured using a NANODROP® 1000 Spectrophotometer. Samples were stored at −80° C.

First strand cDNA was synthesized using a Transcriptor Reverse Transcriptase First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, Ind., USA). Four libraries were prepared for each of the five Aspergillus niger strains described in Example 14 using different quantities of DNase-treated RNA: 300 ng, 600 ng, 900 ng, and 1.2 μg in a volume of 1 to 2 μl combined with 2 μl of random hexamer primer (Roche Applied Science, Indianapolis, Ind., USA) and adjusted to a volume of 13 μl with DEPC-treated water. The sample were incubated for 10 minutes at 65° C. and then placed on ice. A 6.5 μl aliquot of Transcriptor Reverse Transcriptase master mix (4 μl of Transcriptor Reverse Transcriptase buffer, 0.5 μl of Protector RNase inhibitor, and 2 μl of dNTP mix) were added to a tube followed by the addition of a 0.5 μl aliquot of Transcriptor Reverse Transcriptase. The samples were removed from the ice and transferred to an EPPENDORF® MASTERCYCLER® 5333 programmed for 10 minutes at 25° C., 60 minutes at 55° C., and then 5 minutes at 85° C. Samples were stored at −80° C.

Example 16 Detection of Aspergillus niger Polyketide Synthase mRNA by Real-Time PCR

The relative expression levels of Aspergillus niger polyketide synthase mRNA by the Aspergillus niger strains described in Example 14 were quantified by RT-PCR. Total RNA was extracted from each strain to serve as a template for first strand cDNA synthesis as described in Example 15. The first strand cDNA served as a template for RT-PCR. The Aspergillus niger actin gene was used as a reference standard (i.e., internal control). Probes and primers were designed using the Roche Universal ProbeLibrary Design Center Software (Roche Applied Science, Indianapolis, Ind., USA). The following primers and probe pairs were used:

Aspergillus niger polyketide synthase forward primer (62488): (SEQ ID NO: 31) 5′-tcgtgaatcaggtcctagcc-3′ Aspergillus niger polyketide synthase reverse primer (62489): (SEQ ID NO: 32) 5′-aaacaacccaattggtagatgc-3′ Roche Universal ProbeLibrary Probe #80 (04689038001): 5′-cctggaga-3′ Aspergillus niger actin forward primer (62520): (SEQ ID NO: 33) 5′-atctgtacggcaacattgtca-3′ Aspergillus niger actin reverse primer (62521): (SEQ ID NO: 34) 5′-ttctgcatacggtcggagat-3′ Roche Universal ProbeLibrary Probe #131 (04694155001): 5′-ctggtggt-3′

The RT-PCR assay was performed using a LIGHTCYCLER® 480 System and the above Roche Universal ProbeLibrary Probes, which were pre-labeled with fluorescein at the 5′-end and a dark quencher dye near the 3′-end. Each reaction mixture contained 10 μl of LIGHTCYCLER® 480 Probes Master Mix, 0.1 μM Roche Universal ProbeLibrary Probe, 0.2 μM forward primer, 0.2 μM reverse primer, and 2 μl of first strand cDNA (produced as described in Example 15 from varying amounts of total RNA) in a total volume of 20 μl. All RT-PCR reactions were carried out in 384 well plates (Roche Applied Science, Indianapolis, Ind., USA). RT-PCR reactions were carried out in a LIGHTCYCLER® 480 System programmed for 1 cycle of preincubation at 95° C. for 10 minutes, 4.8° C./s; 45 cycles of amplification using the Quantification Analysis mode at 95° C. for 10 seconds, 4.8° C./s; 55° C. for 15 seconds, 2.5° C./s; 72° C. for 1 second, 4.8° C./s (in acquisition mode Single); and 1 cycle of cooling at 40° C. for 10 seconds, 2° C./s. In order to generate standard curves and PCR efficiency values for the target (polyketide synthase gene) and the reference (actin gene), four serial dilutions were created from two cDNA libraries. Each of these serial dilutions was assayed in quadruplicate with both the probe/primer set for the target and reference in separate reactions. The standard curves yielded R² values of 0.996 and 0.998 for the target and reference respectively. The PCR efficiency values calculated by the LIGHTCYCLER® 480 Relative Quantification Software were 1.950 (error 0.0266) and 1.854 (error 0.0181) for the target and reference, respectively. In order to compare the relative level of polyketide synthase mRNA across all five Aspergillus niger strains, three dilutions were tested in triplicate for each of the cDNA libraries. The RT-PCR reactions were carried out using both the probe/primer set for the target and reference in separate reactions for each dilution and replicate. These data were compared with the standard curves using the LIGHTCYCLER® 480 Relative Quantification Software according to the manufacturer. Using this method of analysis, the quantity of expression of the treated sample was calculated relative to the untreated control sample. The quantity of the treated sample was determined from the standard curve divided by the quantity of the untreated control sample. Thus, the untreated sample was designated the 1× sample and all other quantities were expressed as an n-fold difference relative to the untreated sample. Then, the treated sample amount was normalized to an endogenous control, actin, to account for differences in the amount of total RNA added to each reaction.

Using Relative Quantification analysis, the relative expression level of Aspergillus niger polyketide synthase mRNA was determined to be significantly lower in strains pAmFs031-W1 and pAmFs031-W2 compared to strains Aspergillus niger MBin120 pAmFs031-B1, Aspergillus niger MBin120 pDM261, or untransformed Aspergillus niger MBin120, which correlated with the observed changes in spore coloration. The Aspergillus niger MBin120 pAmFs031-W1 and Aspergillus niger MBin120 pAmFs031-W2 strains showed a 68% and 82% reduction in polyketide synthase mRNA compared to the “empty vector” control strain. A Southern blot was performed on the same strains, as described in Example 17, to confirm the presence of the entire polyketide synthase gene and eliminate the possibility that the reduction in transcript was due to a disruption of the polyketide synthase gene itself.

Example 17 Detection of the Aspergillus niger Polyketide Synthase Gene by Southern Blotting

Genomic DNA was extracted from the following Aspergillus niger strains (described in Example 16): untransformed Aspergillus niger MBin120, Aspergillus niger MBin120 pAmFs031-W1, Aspergillus niger MBin120 pAmFs031-W2, Aspergillus niger MBin120 pAmFs031-B1, and Aspergillus niger MBin120 pDM261 using a DNEASY® Plant Maxi Kit. A total of 2 μg of genomic DNA from each strain was digested with Hind III for 17 hours at 37° C. The digested genomic DNA and the DNA molecular weight marker II, DIG-labeled (Roche Applied Science, Indianapolis, Ind., USA) were loaded onto a 0.7% agarose gel in TAE buffer and a current of 22 V was applied for 17 hours. The DNA was transferred from the gel onto a NYTRAN® SuPerCharged membrane for 18 hours using 20×SSC transfer buffer. The DNA was cross-linked to the membrane using ultraviolet irradiation and then equilibrated in DIG Easy Hyb Solution (Roche Applied Science, Indianapolis, Ind., USA) at 42° C. for 30 minutes. The membrane was probed with 20 μl of a DIG-labeled 434 bp PCR product suspended in DIG Easy Hyb solution (Roche Applied Science, Indianapolis, Ind., USA) at 42° C. for 18 hours. The 434 bp DIG-labeled DNA probe was synthesized using a PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, Ind., USA) and the primers shown below.

Aspergillus niger polyketide synthase Southern forward primer (062849): (SEQ ID NO: 35) 5′-ttaattaatcggtcaatcgccgttgtcaga-3′ Aspergillus niger polyketide synthase Southern reverse primer (062850): (SEQ ID NO: 36) 5′-aatttccaaacagggtaactccac-3′

The amplification reaction (50 μl) was composed of 1×PCR Buffer, 5 μl of PCR DIG Probe Synthesis Mix, 50 μM of sense primer, 50 μM of antisense primer, 2.6 units of Expand High Fidelity Polymerase, and 50 ng of Aspergillus niger MBin120 genomic DNA (purified as described above). The reaction was incubated in an EPPENDORF® MASTERCYCLER™ 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 30 seconds at 72° C. (7 minute final extension). The probe sequence is complimentary to the promoter (5′ untranslated region) of the Aspergillus niger polyketide synthase gene, the sequence of which is contained within an 8.8 kb Hind III genomic DNA fragment.

After the membrane was probed, it was washed with 2×SSC plus 0.1% SDS low stringency buffer for 5 minutes at room temperature followed by two high stringency washes with 0.5×SSC plus 0.1% SDS at 65° C. for 15 minutes at room temperature each. The hybridized DIG labeled probe and molecular weight markers were then visualized using a DIG Luminescent Detection Kit (Roche Applied Science, Indianapolis, Ind., USA) following the manufacturer's specifications. The blot was exposed to Biomax XAR film (Sigma Aldrich, St. Louis, Mo., USA) and developed using a Konica SRX-101A film processor (Konica Minolta Medical Imaging USA Inc., Wayne, N.J., USA) to visualize the labeled DNA. The results demonstrated that all of the strains displayed an expected 8.8 kb band indicating that the entire gene was intact in all strains tested.

Example 18 Construction of the Trichoderma reesei Transitive RNAi Expression Vector pAL02

A transitive RNAi expression vector was constructed for suppressing the expression of a Trichoderma reesei beta-xylosidase gene (SEQ ID NO: 37 for the DNA sequence and SEQ ID NO: 38 for the deduced amino acid sequence).

The transitive RNAi plasmid pEvFz-14 was digested with Pac I and Mlu I to isolate the hygromycin inverted repeat. A 527 bp fragment was resolved by 1.0% agarose gel electrophoresis in TAE buffer and then excised from the gel. The fragment was purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The purified fragment was then ligated to Pac I and Mlu I digested pMJ09 (WO 2005/056772) using a Rapid Ligation Kit (Roche Applied Science, Indianapolis, Ind., USA). Two μl of the ligation mixture were used to transform SURE™ chemically competent E. coli cells (Stratagene, La Jolla, Calif., USA) according to manufacturer's instructions. Plasmid DNA from several transformants was purified using a BIOROBOT® 9600 and analyzed by digestion with Pac I and Mlu I. The products of the restriction digest were resolved by 1.0% agarose gel electrophoresis in TAE buffer. One transformant was confirmed by DNA sequence analysis to possess the 527 bp hygromycin inverted repeat and was designated pAL01 (FIG. 8).

PCR was employed to amplify a 500 bp fragment comprising a portion of the Trichoderma reesei beta-xylosidase coding region (SEQ ID NO: 39 for the DNA sequence and SEQ ID NO: 40 for the deduced amino acid sequence) utilizing genomic DNA from Trichoderma reesei RutC30 (WO 2005/056772) serving as template. The genomic DNA from Trichoderma reesei RutC30 was isolated using a DNEASY® Plant Maxi Kit according to the manufacturer's instructions. A sense primer was designed to incorporate a Nco I site at the 5′-end and a reverse primer was designed to incorporate a Mlu I site at the 5′ end as shown below.

Sense primer: 5′-CCATGGTACGAGTTTGGCAGTGGTCT-3′ (SEQ ID NO: 41) Antisense primer: 5′-ACGCGTTTATGCGTCAGGTGTAGCAT-3′ (SEQ ID NO: 42)

The amplification reaction (50 μl) was composed of 10× HERCULASE™ Reaction Buffer (Stratagene, La Jolla, Calif., USA), 0.8 mM dNTPs, 200 ng of Trichoderma reesei RutC30 genomic DNA prepared as described in Example 7, 1 ng of primers, and 2.5 units of HERCULASE HOTSTART™ Polymerase (Stratagene, La Jolla, Calif., USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 92° C. for 2 minutes; 30 cycles each at 92° C. for 30 seconds, 58° C. for 30 seconds, and 68° C. for 1 minute; and a 10 minute final extension. The reaction products were isolated by 1.0% agarose gel electrophoresis in TAE buffer where a 500 bp band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions. The purified PCR fragment was digested with Nco I and Mlu I and ligated using a Rapid Ligation Kit into pAL01 that had been digested with Nco I and Mlu I. The ligation mixture was used to transform SURE™ chemically competent E. coli cells according to the manufacturer's instructions. Then transformants were screened by colony PCR to identify those containing the desired beta-xylosidase insert. The reactions (20 μl) were composed of 2 μl of 10× THERMOPOL™ Reaction Buffer, 0.4 μl of 10 mM dNTPs, 1 μl of an E. coli transformant colony suspended in 50 μl of deionized water, and 1 pmol of the beta-xylosidase amplification primers. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 94° C. for 2 minutes; 17 cycles each at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds; and a 5 minute final extension. The amplification products were isolated by 1.0% agarose gel electrophoresis in TAE buffer. Plasmid DNA from several transformants containing the 500 bp beta-xylosidase amplification product was purified using a BIOROBOT® 9600 and analyzed by DNA sequencing. DNA sequence analysis of one plasmid containing the 500 bp beta-xylosidase amplification product confirmed the expected insertion and was designated pAL02 (FIG. 9).

Example 19 Transformation and Determination of Transitive RNAi in Trichoderma reesei of the Trichoderma reesei Beta-Xylosidase Gene

Five μg of pAL02 was used to transform Trichoderma reesei SaMe13, a cbh1 deleted strain (WO 2005/030926). Trichoderma reesei SaMe13 spores were harvested by pouring 20 ml of 0.01% TWEEN® 80 onto a COVE2 plate of mature Trichoderma reesei SaMe13 and spores were scraped off with a petri dish spreader. The spore mixture was drawn up into a 20 ml pipette. Approximately 2-5×10⁷ spores were inoculated into 100 ml of YP medium supplemented with 2% glucose and 10 mM uridine and incubated at 27° C. at 90 rpm for 16 hours. Mycelia were collected using a 500 ml STERICUP™ filter unit (Millipore, Burlington, Mass., USA) and washed twice with 100 ml of deionized water. The mycelia were then washed twice with 250 ml of 1.2 M sorbitol. The washed mycelia were resuspended in 20 ml of 1.2 M sorbitol, 5 mg of GLUCANEX® per ml and 0.5 mg of chitinase per ml. The mixture was incubated at 34° C. for 15-25 minutes at 90 rpm. The flask was placed on ice for five minutes followed by filtration through MIRACLOTH™. The filter containing the protoplasts was placed into a 50 ml FALCON™ tube (VWR International, West Chester, Pa., USA). The tube was centrifuged at 370×g for 10 minutes in a Sorvall RT6000D centrifuge. The supernatant was discarded and the protoplast pellet was resuspended in 25 ml of 1.2 M sorbitol and centrifuged at 370×g for 10 minutes in a Sorvall RT6000D centrifuge. The supernatant was discarded and pellet resuspended in 25 ml of 1.2 M sorbitol. Ten μl were removed and the tube centrifuged as described above while the protoplasts were counted in a hemocytometer. The supernatant was discarded and the pellet resuspended at a concentration of 1×10⁸ protoplast per ml of STC.

A transformation mixture consisting of 100 μl of the protoplast suspension, 1-10 μg of plasmid DNA in 10 μl of STC, and 250 μl of polyethylene glycol was gently mixed. The mixture was incubated at room temperature for 30 minutes. Three ml of STC was added, mixed, and poured onto a 150 mm COVE plate. The plates were incubated at 28° C. for 10-14 days.

Twenty transformants, AL02-1-AL02-20, and 4 control transformants containing pAL01 were selected and spores from single colonies were streaked onto COVE plates and incubated for 5 days at 28° C. Spores from these plates were inoculated into 125 ml baffled shake flasks containing 25 ml of Cellulase-Inducing Medium and cultivated at 28° C. and 200 rpm for 5 days. One ml samples of culture broth were removed 5 days post-inoculation, centrifuged at 6,000×g for 10 minutes in an EPPENDORF® centrifuge 5415D, and the supernatants transferred to new EPPENDORF® tubes. Duplicates of the broth samples were used to determine beta-xylosidase activity and beta-glucosidase activity.

The supernatants above were assayed for beta-xylosidase activity using a Coulter Biomek 3000, Biomek NX, and a ORCA robotic arm (Beckman Coulter, Inc, Fullerton, Calif., USA). Culture supernatants were diluted appropriately in 0.1 M succinate, 0.01% Triton X-100 pH 5.0 (sample buffer) followed by a series dilution from 0-fold to ⅓-fold to 1/9-fold of the diluted sample. A total of 20 μl of each dilution was transferred to a 96-well flat bottom plate. Two hundred micro-liters of a substrate solution containing 1 mg of para-nitrophenyl-beta-D-xylopyranoside per ml of 0.1 M succinate pH 5.0 was added to each well and then incubated at ambient temperature for 45 minutes. Upon completion of the incubation period, 50 μl of 1 M TRIS buffer pH 9 was added to each well to stop the reaction. An endpoint was measured at an optical density of 405 nm for the 96-well plate.

The supernatants above were assayed for beta-glucosidase activity using a Coulter Biomek 3000, Biomek NX, and a ORCA robotic arm. Culture supernatants were diluted appropriately in 0.1 M succinate, 0.01% Triton X-100 pH 5.0 (sample buffer) followed by a series dilution from 0-fold to ⅓-fold to 1/9-fold of the diluted sample. A total of 20 μl of each dilution was transferred to a 96-well flat bottom plate. Two hundred micro-liters of a substrate solution containing 1 mg of para-nitrophenyl-beta-D-glucopyranoside per ml of 0.1 M succinate pH 5.0 was added to each well and then incubated at ambient temperature for 45 minutes. Upon completion of the incubation period, 50 μl of 1 M TRIS buffer pH 9 was added to each well to stop the reaction. An endpoint was measured at an optical density of 405 nm for the 96-well plate. Sample activity was determined by use of the following equation:

[({OD405/extinction coefficient}×1×10⁶)/incubation time]/sample volume where extinction coefficient=17,749, incubation time=45, and sample volume=0.02.

A ratio between the OD obtained from the beta-xylosidase activity assay and the activity determined from the beta-glucosidase assay was calculated and reported as BX/BG. Since beta-glucosidase occurs natively its activity was used as means to normalize for growth differences between transformants. The results are shown in Table 3.

TABLE 3 Sample Beta-xylosidase¹ Beta-glucosidase² Protein³ Ratio BX/BG⁴ AL02 1 5.53 ± 1.10 965.63 ± 107.02 3.51 ± 0.48 0.006 ± 0.001 AL02 2 10.13 ± 2.38  857.73 ± 40.30  4.23 ± 0.51 0.012 ± 0.004 AL02 3 9.89 ± 1.60 1139.06 ± 37.99  2.54 ± 0.31 0.009 ± 0.002 AL02 4 2.94 ± 0.25 174.90 ± 45.20  1.38 ± 0.25 0.017 ± 0.007 AL02 5 15.67 ± 2.55  919.14 ± 39.01  3.56 ± 0.03 0.017 ± 0.004 AL02 6 15.04 ± 7.12  725.61 ± 225.20 4.58 ± 1.96 0.021 ± 0.004 AL02 7 4.78 ± 0.44 494.99 ± 69.17  3.29 ± 0.17 0.010 ± 0.003 AL02 8 3.87 ± 0.22 507.37 ± 262.44 3.25 ± 0.09 0.008 ± 0.005 AL02 9 9.70 ± 3.52 771.72 ± 35.46  4.25 ± 0.43 0.013 ± 0.005 AL02 10 3.25 ± 1.61 576.40 ± 202.08 2.45 ± 1.04 0.006 ± 0.001 AL02 11 3.47 ± 2.19 667.39 ± 159.68 2.21 ± 0.76 0.005 ± 0.003 AL02 13 7.27 ± 2.44 521.00 ± 66.65  3.40 ± 0.74 0.014 ± 0.004 AL02 14 5.75 ± 2.54 834.19 ± 414.42 4.54 ± 1.58 0.007 ± 0.000 AL02 15 10.58 ± 0.64  625.86 ± 427.59 5.51 ± 2.95 0.017 ± 0.015 AL02 16 1.80 ± 0.60 309.91 ± 39.37  4.51 ± 1.76 0.006 ± 0.002 AL02 17 7.31 ± 6.48 477.29 ± 370.17 6.75 ± 2.44 0.015 ± 0.003 AL02 18 6.04 ± 3.83 556.43 ± 143.08 4.69 ± 1.81 0.011 ± 0.005 AL02 19 1.64 ± 0.42 226.67 ± 24.94  2.49 ± 0.12 0.007 ± 0.001 AL02 20 3.55 ± 1.78 410.71 ± 261.33 3.91 ± 1.97 0.009 ± 0.001 AL03 ev 3.59 ± 0.78 320.76 ± 92.52  3.60 ± 1.36 0.011 ± 0.003 ¹Beta-xylosidase activity was measured using para-nitrophenyl-β-D-xylopyranoside as a substrate. Values are expressed as absorbance units ± standard deviation (n = 2 except for sample AL03 ev for which n = 4). ²Beta-glucosidase activity was measured using p-nitrophenyl-β-glucopyranoside as a substrate. Values are expressed as μmol substrate hydrolyzed per min per ml of culture broth ± standard deviation (n = 2 except for sample AL03 ev for which n = 4). ³Protein concentrations were determined using BCA assay reagents. Values are expressed in mg/ml of culture broth ± standard deviation (n = 2 except for sample AL03 ev for which n = 4). ⁴Ratio of beta-xylosidase and beta-glucosidase activities ± standard deviation (n = 2 except for sample AL03 ev for which n = 4). Approximately 5/20 transformants appeared to express BX/BG with values from 36% to 55% of the control. These results demonstrate that transitive RNAi can be used to successfully knockdown gene expression in Trichoderma reesei.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. 

1-8. (canceled)
 9. A filamentous fungal strain comprising a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to the target gene encoding the biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation relative to each other and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein production of short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, is induced by cultivating the filamentous fungal strain under conditions to produce RNA transcripts of the double-stranded transcribable nucleic acid construct that are then converted to the siRNAs, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the biological substance, and wherein the filamentous fungal strain is an Aspergillus niger strain, Aspergillus oryzae strain, or a Trichoderma reesei strain.
 10. The filamentous fungal strain of claim 9, wherein the first homologous region comprises at least 19 nucleotides of the target gene.
 11. The filamentous fungal strain of claim 9, wherein the second transcribable region with no effective homology to the target gene comprises at least 19 nucleotides.
 12. The filamentous fungal strain of claim 9, wherein the first and second polynucleotides are separated by an intervening sequence.
 13. The filamentous fungal strain of claim 12, wherein the two segments complementary to each other in reverse orientation are separated by a linking sequence.
 14. The filamentous fungal strain of claim 12, wherein expression of the target gene is reduced by at least 20%.
 15. The filamentous fungal strain of claim 12, wherein the interfering RNA interacts with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene.
 16. The filamentous fungal strain of claim 15, wherein expression of the one or more homologues of the target gene is reduced by at least 20%.
 17. A method for producing a biological substance, comprising: (a) cultivating a filamentous fungal strain under conditions conducive for production of the biological substance of interest, wherein the filamentous fungal strain comprises a double-stranded transcribable nucleic acid construct comprising a promoter operably linked to a first polynucleotide comprising a first transcribable region with homology to a target gene encoding an undesirable biological substance and a second polynucleotide comprising a second transcribable region with no effective homology to the target gene, wherein the second transcribable region comprises two segments complementary to each other in reverse orientation relative to each other and the first and second transcribable regions are transcribed as a single mRNA molecule, wherein RNA transcripts of the double-stranded transcribable nucleic acid construct are produced by cultivating the filamentous fungal strain under conditions to produce the RNA transcripts that are then converted to short interfering RNAs (siRNAs), comprising sequences of the target gene to be silenced by the process of transitive RNAi, which interact with RNA transcripts of the target gene to reduce or eliminate expression of the target gene encoding the undesirable biological substance; and wherein the filamentous fungal strain comprises a third polynucleotide encoding the biological substance of interest, and wherein the filamentous fungal strain is an Aspergillus niger strain, Aspergillus oryzae strain, or a Trichoderma reesei strain; and (b) recovering the biological substance of interest from the cultivation medium.
 18. The method of claim 17, wherein the first transcribable region with homology to the target gene comprises at least 19 nucleotides of the target gene.
 19. The method of claim 17, wherein the second transcribable region with no effective homology to the target gene comprises at least 19 nucleotides.
 20. The method of claim 17, wherein the first and second polynucleotides are separated by an intervening sequence.
 21. The method of claim 17, wherein the two segments complementary to each other in reverse orientation are separated by a linking sequence.
 22. The method of claim 17, wherein expression of the target gene is reduced by at least 20%.
 23. The method of claim 17, wherein the interfering RNA interacts with RNA transcripts of one or more homologues of the target gene to reduce or eliminate expression of the one or more homologues of the target gene.
 24. The method of claim 23, wherein expression of the one or more homologues of the target gene is reduced by at least 20%. 